Retroviruses are important viral pathogens of animals and humans, causing a variety of different diseases including cancer and AIDS. Most of these viruses including the gammaretrovirus, murine leukemia virus (MLV), can efficiently infect dividing, but not non-dividing, cell types. By contrast other retroviruses, such as the lentivirus HIV-1, can efficiently infect both dividing and non-dividing cells. We hypothesize that virus- specific interactions with intracellular host cell factors are responsible, at least in part, for this difference in target cell tropism between these retroviruses. To examine this hypothesis we are identifying and characterizing cellular factors which regulate infection by MLV, but not by HIV-1. By performing somatic cell- based mutagenesis studies, as well as the first comprehensive genome-wide siRNA screen of its type, we have made the remarkable observation that there are several hundred novel intracellular factors required for infection by MLV, but not by HIV-1. These findings demonstrate that retroviruses interact with many more cellular factors than was thought previously. The factors involved include ZASC1, a protein containing multiple zinc finger regions, as well as PAPSS1 and PAPSS2, two biosynthetic enzymes that synthesize 3' phosphoadenosine 5' phosphosulfate (PAPS), the high energy sulfate donor used in all sulfation reactions in the cell. The goals of the studies in this proposal include examining the mechanisms of action of ZASC1, and the PAPSS enzymes during MLV infection. We will also determine the steps during MLV replication that are regulated by each of the other cellular factors identified and establish whether these factors also regulate infection by other gammaretroviruses including the pig endogenous retroviruses (PERVs). This research will contribute substantially to our understanding of the roles played by cellular factors during retrovirus replication and should give new insights into why gammaretrovirus infection is restricted only to dividing cell types. Moreover, because of the risk of PERV transmission to humans, during pig to human xenotransplantations, these studies are of immediate medical relevance since they could suggest novel antiviral approaches aimed at disrupting critical gammaretrovirus-host cell factor interactions.